Meiosis reduces the normal cellular diploid chromosome content by half to create haploid gametes. Diploid chromosome homologs must be paired prior to this reduction division. Pairing involves the introduction of several hundred DNA double stranded breaks (DSBs) throughout the chromosomes, which then use the cognate chromosome homolog to repair and reassemble individual chromosome pairs. DSB repair requires the RAD51 recombinase to identify homology and perform strand exchange between homologous chromosomes that results in a D-loop: the progenitor to a Holliday Junction (HJ) crossover. D-loop progenitor HJ intermediates are recognized by the meiosis-specific MutS homologs (MSH) MSH4-MSH5, which form ATP-bound sliding clamps that embraces both the participating duplex DNA strands; stably linking the homologous chromosomes. The MutL homologs (MLH) MLH1-MLH3 specifically interact with MSH4-MSH5 and ultimately appear to determine which of the DSB repair events results in genetic crossing-over. This seemingly risky but generally accurate DSB repair progression performs two tasks: I.) the robust pairing of homologous chromosomes prior to spindle formation and meiosis I segregation, and II.) The reassortment of genetic information that is the basis of modern genetics. Mistakes in meiosis chromosome pairing and segregation are the frequent cause of spontaneous miscarriages as well as genetic diseases such as Down syndrome (Trisomey 21) the cooperative interactions between RAD51, MSH4-MSH5 and MLH1-MLH3 is unknown. However, these interactions are likely to be substantial since the D-loop intermediates catalyzed by RAD51 are unstable when RAD51 is removed. Moreover, chromosome pairing is absent when MSH4 or MSH5 are mutated and chromosome segregation does not occur properly when MLH1 or MLH3 are absent; leading to a lack of viable gametes. The goal of this exploratory proposal is to develop new and quantitative probes to understand the complex interactions between RAD51, MSH4-MSH5 and MLH1-MLH3 that result in chromosome pairing during meiosis I. We have developed three robust single molecule measures capable of interrogating and visualizing the functions of these essential meiosis I components in real-time. We propose two specific aims: 1.) analysis of the interaction between RAD51 and MSH4-MSH5 during recombination mediated chromosome pairing, and 2.) analysis of the interactions between MSH4-MSH5, MLH1-MLH3 and Holliday Junctions. We appear to be the only group examining the ensemble functions between these essential meiosis chromosome-pairing components. Our unique single molecule approach should enhance the quantitative understanding of mechanical processes that lead to viable gamete formation as well as the fine line between dysfunctions that lead to infertility and genetic disease.